Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. My current research uses the genetically-tractable bacterium ''Bacillus subtilis'' as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. I have been characterizing the protein ConE (formerly YddE) which is encoded on the ''B. subtilis'' conjugal element ICE''Bs1''. ConE is related to proteins encoded on conjugal elements in numerous bacteria, including the Gram-positive pathogens ''S. aureus, C. difficile'', and ''L. monocytogenes''. ConE belongs to a large superfamily of ATP-dependent pumps involved in the extrusion of proteins and DNA through membrane pores. I have shown that ConE and its ATPase domain are essential for mating of ICE''Bs1''. In addition, ConE-GFP localizes at the cell poles, in close association with the membrane (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, I propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. I plan on analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques.

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Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. We use the genetically-tractable bacterium ''Bacillus subtilis'' as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. We have been characterizing the protein ConE (formerly YddE) which is encoded on the ''B. subtilis'' conjugative element ICE''Bs1''. ConE is related to proteins encoded on conjugative elements in numerous bacteria, including the Gram-positive pathogens ''S. aureus, C. difficile'', and ''L. monocytogenes''. ConE belongs to a large superfamily of ATP-dependent pumps, such as VirB4, FtsK, and SpoIIIE, involved in the extrusion of proteins and DNA through membrane pores. We have shown that ConE and its ATPase domain are essential for mating of ICE''Bs1''. In addition, ConE-GFP localizes at the membrane, predominantly at the cell poles (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, we propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. We are analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques. Our research is funded through Suffolk University and an NSF-RUI grant from 2012-2015.

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== Current Members of the Berkmen Lab ==

== Current Members of the Berkmen Lab ==

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[[Image: Steph_Bridget_Sci_Banq_2010.JPG |300px|thumb|Stephanie and Bridget at their poster at the Suffolk University Science Banquet, Spring 2010|right]]

Matt joined out lab under the NSF grant in the summer of 2012 as a research assistant. He has explored the role of ''yddF'' in mating and will soon be exploring the determinants of localization of ConE-GFP. <br>

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<br>

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'''Gianna Mancuso ''' - Biochemistry Major<br>

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Gianna has been working as a research assistant since the spring of 2011. She, along with Kyle Swerdlow, helped optimize the purification of His6-ConE and the research was presented at the 2012 ACS national conference and 2012 STEM conference at Suffolk University. She has also helped clone ConE fusions for use in bacterial two-hybrid.

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'''Bridget Giarusso''' - Biochemistry Major<br>

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'''Georgeanna Morton''' - Biochemistry Major<br>

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As a research assistant, Bridget and Stephanie used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. Since His6-ConE is functional for mating, we can purify His6-ConE and begin ''in vitro'' assays to determine whether it can bind ATP. She presented this research with Stephanie at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She is also helping to clone several ICE''Bs1'' genes.<br>

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Geogeanna became a research assistant under the NSF grant in the summer of 2012. For her senior thesis, she is purifying His6-ConE mutants and testing the oligomerization of each using Blue Native-PAGE. <br>

<br>

<br>

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'''Stephanie Laurer''' - Biochemistry Major<br>

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As a research assistant, Stephanie and Bridget used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. Since His6-ConE is functional for mating, we can purify His6-ConE and begin ''in vitro'' assays to determine whether it can bind ATP. She presented this research with Bridget at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She is also helping to clone several ICE''Bs1'' genes. On the side, Stephanie has had an interest in genetically modified food. During her first year at Suffolk, she used a PCR-based assay to detect the Bt gene in corn. She found that at least half of the samples she tested (6 of 11) were genetically modified. She presented this work at the 2009 Suffolk science banquet where her poster won first place.<br>

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'''Azul Pinochet Barros''' - Biology/Philosophy Major<br>

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Azul joined the lab in the spring of 2012. With an interest in cyanobacteria, Azul was very determined to be involved in bacterial growth, so she has helped optimize our competent cells procedure. <br>

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<br>

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'''Kyle Swerdlow''' - Biochemistry Major<br>

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Kyle started working as a research assistant in fall of 2011 working very closely with Gianna Mancuso on the optimization of His6-ConE purification. They presented this research at the 2012 ACS national conference as well as the 2012 STEM conference at Suffolk University. Kyle is now using bacterial two-hybrid analysis to piece together key players in the ICEBs1 conjugation machinery. <br>

Stephanie used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. She presented this research with Bridget at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She also helped clone several ICE''Bs''1 genes. On the side, Stephanie had an interest in genetically modified food. During her first year at Suffolk, she used a PCR-based assay to detect the Bt gene in corn. She found that at least half of the samples she tested (6 of 11) were genetically modified. She presented this work at the 2009 Suffolk science banquet where her poster won first place. She now acts as Clinical Research Assistant at Beth Israel Deaconess Medical Center.<br>

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<br>

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'''Bridget Giarusso''' - B.S. Biochemistry, May 2011<br>

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As a research assistant, Bridget used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. She presented this research with Stephanie at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She helped clone several ICE''Bs''1 genes.<br>

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<br>

'''Matt Hamada''' - B.S. Biochemistry, December 2010<br>

'''Matt Hamada''' - B.S. Biochemistry, December 2010<br>

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For CHEM L333, Matt cloned the ''yddC'' gene encoded on the ICE''Bs1'' conjugal element. For CHEM L428/L429, Matt will be using fluorescence microscopy to determine whether ''yddC'' and other ICE''Bs1'' genes are required for ConE to localize at the cell poles. He presented his research with Cori at the 2009 Boston Bacterial Meeting.<br>

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For CHEM L333, Matt cloned the ''conC'' gene encoded on the ICE''Bs1'' conjugative element. For CHEM L428/L429, Matt used fluorescence microscopy to determine whether ''conC'' and other ICE''Bs1'' genes are required for ConE to localize at the cell poles. He presented his research with Cori at the 2009 Boston Bacterial Meeting. Currently, Matt is working as a laboratory research technician.<br>

<br>

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'''Cori Leonetti''' - B.S. Biochemistry, May 2010<br>

'''Cori Leonetti''' - B.S. Biochemistry, May 2010<br>

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For CHEM L333, Cori helped clone the ''yddB'' gene encoded on the ICE''Bs1'' conjugal element. Cori is extending her CHEM L333 project for CHEM L428/L429. She will be testing whether ''yddB'' and other ICE''Bs1'' genes are required for mating. Cori presented her research with Matt at the 2009 Boston Bacterial Meeting.<br>

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For CHEM L333, Cori helped clone the ''conB'' gene encoded on the ICE''Bs1'' conjugative element. Cori extended her CHEM L333 project for CHEM L428/L429. She tested whether ''conB'' and other ICE''Bs1'' genes are required for mating. Cori presented her research with Matt at the 2009 Boston Bacterial Meeting. Cori is now a graduate student at Arizona State University studying microbiology. <br>

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'''Erin Cross''' - B.S. Biochemistry, May 2009<br>

'''Erin Cross''' - B.S. Biochemistry, May 2009<br>

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In CHEM L333, Erin and her class mates attempted to clone various C-terminal truncations of YddE fused to GFP. For CHEM L428/L429, she used fluorescence microscopy to analyze what parts of YddE are required for localization to the cell poles. She found that the C-terminal half of YddE is critical for localization. In addition, she found the YddE-GFP localizes at the cell poles, even when the upstream gene ''yddD'' is not expressed ''in cis''. She presented this work at the national ACS meeting in March 2009 and at the 2009 Suffolk Science Banquet where her poster won 3rd place.<br>

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In CHEM L333, Erin and her class mates attempted to clone various C-terminal truncations of ConE fused to GFP. For CHEM L428/L429, she used fluorescence microscopy to analyze what parts of ConE are required for localization to the cell poles. She found that the C-terminal half of ConE is critical for localization. She presented this work at the national ACS meeting in March 2009 and at the 2009 Suffolk Science Banquet where her poster won 3rd place. Erin is now working as a laboratory research technologist at MMCRI.<br>

For CHEM L333, Maria began construction of a his-tagged YddE. For CHEM L428/L429, she purified and characterized His6-YddE to enable future students to perform ATPase assays to determine whether YddE can hydrolyze ATP ''in vitro''. She presented this work at the national ACS meeting in March 2009.<br>

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For CHEM L333, Maria constructed a his-tagged ConE. For CHEM L428/L429, she purified and characterized His6-ConE to enable future students to perform ATPase assays to determine whether ConE can hydrolyze ATP ''in vitro''. She presented this work at the national ACS meeting in March 2009. Maria is now a graduate student in the molecular and cellular biology Ph.D. program at Ohio University.<br>

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'''Tamara Wong''' - B.S. Biochemistry Forensic Science, May 2009<br>

'''Tamara Wong''' - B.S. Biochemistry Forensic Science, May 2009<br>

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Tamara cloned the His6-YddE construct so that we can test whether this protein can support mating. In addition, Tamara purified His6-YddE to enable future ATPase assays.<br>

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Tamara helped cloned the His6-ConE construct so that we can test whether this protein can support mating. In addition, Tamara purified His6-ConE to enable future ATPase assays.<br>

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'''Emma-Kate Loveday''' - B.S. Biochemistry, May 2008<br>

'''Emma-Kate Loveday''' - B.S. Biochemistry, May 2008<br>

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For her CHEM L428/L429 project, Emma-Kate constructed two variants of YddE and tested their effects on mating. She found that the Walker B (ATP hydrolysis domain) of YddE is essential for mating. She also found that the N-terminus of YddE does not contribute significantly to mating. She presented her work at the Boston Bacterial Meeting in June 2008 and the Cold Spring Harbor Molecular Genetics of Bacteria and Phages Meeting in August 2008. Emma is now a graduate student in the microbiology and immunology Ph.D. program at the University of British Columbia supported by a prestigious NSF Fellowship.<br>

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For her CHEM L428/L429 project, Emma-Kate constructed two variants of ConE and tested their effects on mating. She found that the Walker B (ATP hydrolysis domain) of ConE is essential for mating. She also found that the N-terminus of ConE does not contribute significantly to mating. She presented her work at the Boston Bacterial Meeting in June 2008 and the Cold Spring Harbor Molecular Genetics of Bacteria and Phages Meeting in August 2008. Emma is now a graduate student in the microbiology and immunology Ph.D. program at the University of British Columbia supported by an NSF Fellowship.<br>

Berkmen MB and Grossman AD. (2007) Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA. Mol Microbiol, 63(1): 150-165.

'''Berkmen MB''' and Grossman AD. (2007) Subcellular positioning of the origin region of the ''Bacillus subtilis'' chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA. Mol Microbiol, 63(1): 150-165.

My husband, Mehmet Berkmen, is also a microbiologist. He was a postdoctoral fellow in [http://beck2.med.harvard.edu/ Jon Beckwith's lab] at Harvard Medical School.<br>

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In my free time I like to travel, snowboard, practice my Turkish, and eat delicious food.

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Now he is at the biotechnology company [http://www.neb.com/nebecomm/default.asp New England Biolabs], where he is developing ''Escherichia coli'' strains and plasmids for recombinant protein production.<br>

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<br>

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In my free time, I like to travel, snowboard, practice my Turkish, and eat delicious food.

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[[Image:Kaan_0108_redsweater.jpg|300px|thumb|My son Kaan at 1 year|left]]<br>

Bacterial mating or conjugation is the transfer of DNA from one bacterium to another via direct cell-to-cell contact through a mating pore. We use the genetically-tractable bacterium Bacillus subtilis as a model system to explore the function and subcellular localization of a putative component of the bacterial mating pore apparatus. We have been characterizing the protein ConE (formerly YddE) which is encoded on the B. subtilis conjugative element ICEBs1. ConE is related to proteins encoded on conjugative elements in numerous bacteria, including the Gram-positive pathogens S. aureus, C. difficile, and L. monocytogenes. ConE belongs to a large superfamily of ATP-dependent pumps, such as VirB4, FtsK, and SpoIIIE, involved in the extrusion of proteins and DNA through membrane pores. We have shown that ConE and its ATPase domain are essential for mating of ICEBs1. In addition, ConE-GFP localizes at the membrane, predominantly at the cell poles (see Figure). Given ConE’s localization, ATPase domain, and essentiality in conjugation, we propose that ConE and its homologs are the essential membrane-associated ATPase component of the Gram-positive mating pore apparatus. We are analyzing the role of ConE in conjugation, exploring its functional domains, and investigating its subcellular localization through a combination of bioinformatics, molecular, cellular, and biochemical techniques. Our research is funded through Suffolk University and an NSF-RUI grant from 2012-2015.

Current Members of the Berkmen Lab

Matt Broulidakis - Biochemistry Major, Honors Program
Matt joined out lab under the NSF grant in the summer of 2012 as a research assistant. He has explored the role of yddF in mating and will soon be exploring the determinants of localization of ConE-GFP.

Gianna Mancuso - Biochemistry Major
Gianna has been working as a research assistant since the spring of 2011. She, along with Kyle Swerdlow, helped optimize the purification of His6-ConE and the research was presented at the 2012 ACS national conference and 2012 STEM conference at Suffolk University. She has also helped clone ConE fusions for use in bacterial two-hybrid.

Georgeanna Morton - Biochemistry Major
Geogeanna became a research assistant under the NSF grant in the summer of 2012. For her senior thesis, she is purifying His6-ConE mutants and testing the oligomerization of each using Blue Native-PAGE.

Azul Pinochet Barros - Biology/Philosophy Major
Azul joined the lab in the spring of 2012. With an interest in cyanobacteria, Azul was very determined to be involved in bacterial growth, so she has helped optimize our competent cells procedure.

Kyle Swerdlow - Biochemistry Major
Kyle started working as a research assistant in fall of 2011 working very closely with Gianna Mancuso on the optimization of His6-ConE purification. They presented this research at the 2012 ACS national conference as well as the 2012 STEM conference at Suffolk University. Kyle is now using bacterial two-hybrid analysis to piece together key players in the ICEBs1 conjugation machinery.

Former Members of the Berkmen Lab

Stephanie Laurer - B.A. Biochemistry, Honors Program, May 2012
Stephanie used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. She presented this research with Bridget at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She also helped clone several ICEBs1 genes. On the side, Stephanie had an interest in genetically modified food. During her first year at Suffolk, she used a PCR-based assay to detect the Bt gene in corn. She found that at least half of the samples she tested (6 of 11) were genetically modified. She presented this work at the 2009 Suffolk science banquet where her poster won first place. She now acts as Clinical Research Assistant at Beth Israel Deaconess Medical Center.

Bridget Giarusso - B.S. Biochemistry, May 2011
As a research assistant, Bridget used mating assays to determine that addition of a His6-tag on the N-terminus of ConE does not interfere with ConE's ability to support mating. She presented this research with Stephanie at the 2010 ACS National Meeting in San Francisco and at the Suffolk Science Banquet where they won a poster award. She helped clone several ICEBs1 genes.

Matt Hamada - B.S. Biochemistry, December 2010
For CHEM L333, Matt cloned the conC gene encoded on the ICEBs1 conjugative element. For CHEM L428/L429, Matt used fluorescence microscopy to determine whether conC and other ICEBs1 genes are required for ConE to localize at the cell poles. He presented his research with Cori at the 2009 Boston Bacterial Meeting. Currently, Matt is working as a laboratory research technician.

Cori Leonetti - B.S. Biochemistry, May 2010
For CHEM L333, Cori helped clone the conB gene encoded on the ICEBs1 conjugative element. Cori extended her CHEM L333 project for CHEM L428/L429. She tested whether conB and other ICEBs1 genes are required for mating. Cori presented her research with Matt at the 2009 Boston Bacterial Meeting. Cori is now a graduate student at Arizona State University studying microbiology.

Erin Cross - B.S. Biochemistry, May 2009
In CHEM L333, Erin and her class mates attempted to clone various C-terminal truncations of ConE fused to GFP. For CHEM L428/L429, she used fluorescence microscopy to analyze what parts of ConE are required for localization to the cell poles. She found that the C-terminal half of ConE is critical for localization. She presented this work at the national ACS meeting in March 2009 and at the 2009 Suffolk Science Banquet where her poster won 3rd place. Erin is now working as a laboratory research technologist at MMCRI.

Maria Muccioli (formerly Levicheva) - B.S. Biochemistry, Honors Program, May 2009
For CHEM L333, Maria constructed a his-tagged ConE. For CHEM L428/L429, she purified and characterized His6-ConE to enable future students to perform ATPase assays to determine whether ConE can hydrolyze ATP in vitro. She presented this work at the national ACS meeting in March 2009. Maria is now a graduate student in the molecular and cellular biology Ph.D. program at Ohio University.

Tamara Wong - B.S. Biochemistry Forensic Science, May 2009
Tamara helped cloned the His6-ConE construct so that we can test whether this protein can support mating. In addition, Tamara purified His6-ConE to enable future ATPase assays.

Emma-Kate Loveday - B.S. Biochemistry, May 2008
For her CHEM L428/L429 project, Emma-Kate constructed two variants of ConE and tested their effects on mating. She found that the Walker B (ATP hydrolysis domain) of ConE is essential for mating. She also found that the N-terminus of ConE does not contribute significantly to mating. She presented her work at the Boston Bacterial Meeting in June 2008 and the Cold Spring Harbor Molecular Genetics of Bacteria and Phages Meeting in August 2008. Emma is now a graduate student in the microbiology and immunology Ph.D. program at the University of British Columbia supported by an NSF Fellowship.

Berkmen MB and Grossman AD. (2007) Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA. Mol Microbiol, 63(1): 150-165.